Method for spatially high-resolution luminescence microscopy of a sample which is labelled with label molecules which can be activated by a switching signal such that they can be excited to emit particular luminescence radiation only in the activated state, wherein the method has the following steps:    a) introducing the switching signal onto the sample such that only a subset of the label molecules present in the sample are activated, wherein there are area parts in the sample in which activated label molecules have a distance to their closest neighbouring activated labelled molecules that is at least greater than or equal to a length which results from a predetermined optical resolution,    b) exciting the activated molecules to emit luminescence radiation,    c) detecting the luminescence radiation with the predetermined optical resolution and    d) generating a frame from the luminescence radiation recorded in step c), wherein the geometric locations of the label molecules emitting luminescence radiation are identified with a spatial resolution increased above the predetermined optical resolution,
wherein the steps are repeated several times and the several thus-obtained frames are combined into a complete image.
A standard field of use of light microscopy for examining biological preparations is luminescence microscopy. In this process, particular dyes (so-called phosphors or fluorophores) are used for the specific labelling of samples, e.g. of cell parts. The sample is, as mentioned, illuminated with illumination radiation realizing excitation radiation and the luminescence radiation excited thereby is recorded by suitable detectors. For this, a dichroic beam splitter is usually provided in the microscope in combination with block filters which split the luminescence radiation from the excitation radiation and enable an independent observation. Through this procedure, the imaging of individual, differently coloured cell parts is possible with the microscope. Of course, several parts of a preparation can also be simultaneously coloured with different dyes attaching specifically to different structures of the preparation. This method is called multiple luminescence. Samples which luminesce per se, thus without added dye, can also be surveyed.
Here, luminescence is understood, as is generally usual, as a generic term for phosphorescence and fluorescence, thus covers both processes. When fluorescence is mentioned here, it is to be understood pars pro toto and not to be limiting.
To examine samples, it is also known to use laser scanning microscopes (also LSM for short) which, from a three-dimensionally illuminated image, image by means of a confocal detection arrangement (when it is called a confocal LSM) or a non-linear sample interaction (so-called multiphoton microscopy) only that plane which is located in the focal plane of the objective. An optical section is produced and the recording of several optical sections at different depths of the sample then allows the generation, with the help of a suitable data-processing device, of a three-dimensional image of the sample which is composed of the different optical sections. Laser scanning microscopy is thus suitable for examining thick preparations.
Of course, a combination of luminescence microscopy and laser scanning microscopy is also used, in which a luminescent sample is imaged at different depth planes with the help of an LSM.
In principle, the optical resolution of a light microscope, also that of an LSM, is diffraction-limited by physical laws. For the optimum resolution within these limits, specific illumination configurations are known, such as for example a 4Pi arrangement or arrangements with standing-wave fields. Then, the resolution can be clearly improved, in particular in axial direction, over that of a standard LSM. Using non-linear depopulation processes, the resolution can be further increased to a factor of up to 10 compared with a diffraction-limited confocal LSM. Such a method is described for example in U.S. Pat. No. 5,866,911. Different approaches are known for the depopulation processes, for example as described in DE 4416558 C2, U.S. Pat. No. 6,633,432 or DE 10325460 A1.
A further method for increasing resolution is discussed in EP 1157297 B1. There, non-linear processes are utilized by means of structured illumination. The document mentions the saturation of the fluorescence as non-linearity. The described method claims to realize a shift of the object space spectrum relative to the transmission function of the optical system through a structured illumination. Specifically, the shift of the spectrum means that object space frequencies V0 are transmitted at a spatial frequency V0-Vm, wherein Vm is the frequency of the structured illumination. At a given spatial frequency maximally transmissible by the system, this enables the transfer of spatial frequencies of the object exceeding the maximum frequency of the transmission function by the shift frequency Vm. This approach requires a reconstruction algorithm for image generation and the utilization of several frames for an image. In this method also, it is to be considered disadvantageous that the sample is unnecessarily stressed with radiation in areas outside the detected focus, as the necessary structured illumination covers the whole sample volume. Moreover, this method cannot currently be used in the case of thick samples, as extra-focally excited fluorescence also reaches the detector as a background signal and thus dramatically reduces the dynamic range of the detected radiation.
A method which, independently of laser scanning microscopy, achieves a resolution beyond the diffraction limit is known from WO 2006127692 and DE 102006021317. This method, PALM for short, (Photo Activated Light Microscopy) uses a label substance which can be activated by means of an optical activation signal. Only in the activated state can the label substance be excited by excitation radiation to emit particular fluorescence radiation. Non-activated molecules of the label substance also emit after irradiation by excitation radiation no, or at least no noticeable, fluorescence radiation. The activation radiation thus switches the label substance into a state in which it can be excited to fluorescence. Different activation, e.g. of thermal type, is also possible. Therefore, the general term switching signal is used. In the PALM method, the switching signal is applied such that at least a certain proportion of the activated label molecules are spaced apart from neighbouring activated molecules such that they are separated, as measured by the optical resolution of the microscopy, or can be separated subsequently. The activated molecules are thus at least largely isolated. After recording the luminescence radiation, for these isolated molecules the centre of their radiation distribution caused in resolution-limited manner is then identified and the position of the molecules computationally determined from it with higher precision than the optical imaging itself allows. This increased resolution by computational determination of the centre of the diffraction distribution is also called “superresolution” in the state of the art. It requires at least some of the activated label molecules to be distinguishable, thus isolated, in the sample with the optical resolution with which the luminescence radiation is detected. For such molecules, the location information can then be achieved with increased resolution.
To isolate individual label molecules, the PALM method exploits the fact that the probability of a label molecule being activated after receipt of the switching signal of given intensity, e.g. a photon of the activation radiation, is the same for all molecules. Via the intensity of the switching signal and thus the number of photons which strike a unit area of the sample, it is thus possible to ensure that the probability of activating label molecules present in a given unit area of the sample is so small that there are enough areas in which only distinguishable label molecules emit fluorescence radiation within the optical resolution. The result of a suitable choice of the intensity, e.g. of the photon density, of the switching signal, is that, as far as possible, only label molecules isolated relative to the optical resolution are activated and subsequently emit fluorescence radiation. For these isolated molecules, the centre of the intensity distribution conditional on diffraction and thus the location of the label molecule is then identified computationally with increased resolution. To image the whole sample, the isolation of the label molecules of the subset is repeated by introducing activation radiation, subsequent excitation and fluorescence radiation imaging until, if possible, all label molecules were contained once in a subset and isolated within the resolution of the imaging.
The PALM method has the advantage that a high local resolution is necessary for neither the activation nor the excitation. Instead, both the activation and the excitation can be effected in wide-field illumination.
As a result, the label molecules are statistically activated in partial quantities by suitable choice of the intensity of the activation radiation. Therefore, a plurality of frames must be evaluated for the generation of a complete image of a sample in which the locations of all label molecules can be determined computationally with e.g. resolution lying beyond the diffraction limit. There can be up to 10,000 frames. The result of this is that large data quantities are processed, and the measurement lasts a correspondingly long time. The acquisition of a complete image alone requires several minutes, which is fixed essentially by the read-out rate of the camera used. The determination of the position of the molecules in the frames takes place by elaborate computational procedures, as described for example in Egner et al., Biophysical Journal, pp. 3285-3290, volume 93, November 2007. The processing of all frames and their combination into a high-resolution complete image, thus an image in which the locations of the label molecules are given with a resolution lying beyond the diffraction limit, typically lasts four hours.